Model experiments show that disruption of particular multidrug resistance (MDR) genes in Escherichia coli and Staphylococcus aureus increases sensitivity to a variety of antimicrobials including natural compounds. This increased sensitivity exhibited by MDR- knockout mutants results in a potentially more effective screening format for the detection of new antimicrobial compounds present in low concentrations in natural product extract libraries. An E. coli mutant was used in a pilot study to find a number of antimicrobials that were not detected with the corresponding wild-type strain. We propose to design an S. pneumoniae strain essentially devoid of MDR pumps and use it for drug screening. The choice of organism was based on the following criteria: its clinical significance as an important human pathogen; emerging drug resistance; the clinical need to develop new antibiotics directed against Gram positive pathogens; the definition and public availability of the complete genome sequence that allows for an assignment of potential MDR genes followed by PCR amplification and gene disruption. There are four putative MDR pumps in S. pneumoniae. After MDR-gene disruption, the MDR-less mutant will be used for screening large libraries of extracts from plants and marine organisms to discover new and efficacious antimicrobials. PROPOSED COMMERCIAL APPLICATION: Drug resistant bacterial pathogens are a growing and significant health problem. There is a dire medical need to discover and develop new antibiotics. The research described herein will lead to establishing a new technology for sensitive screening of natural product libraries to identify new antimicrobials. In the course of this project, screening of natural product libraries will produce new antibacterial lead compounds for drug development.